Many methods and compositions are known for calibrating mass spectrometers. As explained by Moini in Rapid Communications in Mass Spectrometry 8:711-714(1994), use of calibration compounds which generate highly accurate mass reference peaks is critically important in mass spectrometry, insofar as any error introduced by a calibrant can be magnified and cause a significant loss in accuracy.
The compounds and compositions used to calibrate mass spectrometers vary widely. Many of the known calibrating compositions have a number of limitations. For example, most calibrants are useful in calibrating only certain ranges of a mass spectrum but are not useful across a broad range of mass-to-charge (m/z) values. Many calibrants are useful in electrospray mass spectrometry but cannot be used in APCI mass spectrometry and vice versa, or may be limited to use in only one ion polarity. Furthermore, some calibrants may include significant isotopic contributions (i.e., .sup.13 C) with increasing mass-to-charge or may be multiply charged limiting their effectiveness as reference masses. Additional shortcomings may include rapid ion source contamination, loss of sensitivity, or a combination of factors.
Several references are of interest insofar as they pertain to calibration compounds and/or methods for use in mass spectrometry. U.S. Pat. No. 4,260,886 to Grilletto et al., for example, describes a method for improving the accuracy of a spectrometric measurement of a small volume of gas by carefully monitoring the volume and injection rate of the calibrating gas. U.S. Pat. No. 5,401,664 to Larson et al. describes a ratiometric calibration method for determining the concentration of dibutyl and monobutyl phosphate degradation products resulting from a solvent extraction process, the method involving the use of a labeled internal standard comprising the deuterated phosphates of interest and bistrimethylsilyltrifluoracetamide as a silylating agent. U.S. Pat. No. 4,847,493 to Sodal et al. describes a calibration assembly for use in calibrating a mass spectrometer and designed to reduce the amount of calibration gas and time that is used in the calibration process. U.S. Pat. No. 5,051,583 to Mimura et al. describes an atmospheric pressure ionization type mass spectrometer in which a mass marker of the mass spectrometer is derived using water cluster ions accelerated by a drift voltage applied between the ionization chamber of the spectrometer and the spectrometer's mass analysis region.
Additional references of interest are Stout et al., Organic Mass Spectrometry 25:187-190 (1990), Saar et al., Organic Mass Spectrometry 26:660-663 (1991), and Stout et al., Anal. Chem. 61:2126-2128 (1989), which relate to tuning and calibration in thermospray liquid chromatography/mass spectrometer systems using perfluorinated alkyl acids, ammonium salts of perfluorinated alkyl acids (Stout et al. (1990)), acetic acid cluster ions (Saar et al.), and trifluoroacetic acid cluster ions (Stout et al. (1989)). Moini, supra, describes the use of "Ultramark.RTM. 1621" as a calibration/reference compound in electrospray mass spectrometry. Ultramark.RTM. 1621 (available from PCR, Gainesville, Fla.) is a mixture of fluorinated phosphazines, with each phosphazine randomly and heterogeneously substituted, i.e., the various substituents bound to the phosphorus atoms in the heterocyclic phosphazine ring are each different (note that the compounds referred to in Moini as "phosphazines" are referred to herein as "triazatriphosphorines"). While the mixture is somewhat effective, the Moini mixture is useful only in the mass range of approximately m/z 900 to m/z 2100; the mixture is not useful across a broad range of m/z values. The mass spectra obtained with the Moini heterogeneous mixture are characterized by a Gaussian distribution of mass peaks approximately 100 amu apart, a distribution which is in general undesirable since the "low end" and "high end" ions are significantly less intense than those which are mid-spectrum. Finally, the mixture described by Moini calls for a relatively high concentration of Ultramark.RTM. 1621 (0.05%) which can result in increased background signal (i.e., memory effects).
The present invention addresses each of these limitations in the prior art by providing a calibrant composition which is useful across a broad spectral range, can be used in both electrospray mass spectrometry and in APCI mass spectrometry, and is useful in both positive-ionization and negative-ionization modes. The composition utilizes singly charged compounds which provide for more accurate mass measurements, not just with singly charged species but with multiply charged species (e.g., proteins) as well. The compounds are highly sensitive, nontoxic, non-odorous, relatively low cost, and stable in solution at room temperature for on the order of a year or more.